Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54346—Nanoparticles
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to an efficient and inexpensive method and a test kit for the detection of analytes in a sample.
• biological structures are all molecules that forms of organisms ge ⁇ be absorbed or released; chemical structures are understood to mean all compounds which are able to interact with other molecules in such a way that their detection is possible.
• a sample is a good or a part or a small amount of it, the quality of which is to be tested physically, chemically and / or biologically.
• Biological samples are, for example, part or a small amount of serum, blood, urine, breathing air, tear fluid or the like.
• samples according to the invention are also partial portions taken from waste water, industrial process residues, bogs or other environmental liquids.
• analytes to be examined can be, for example, proteins, peptides, nucleic acids, sequence segments, carbohydrates, lipids and / or antigenic structures.
• the informative value of a parameter investigation can be expanded and improved by parallel acquisition of a larger amount of data from a single sample - the so-called multi-parameter analysis or multi-ligand analysis.
• the parallel acquisition also requires, for example, miniaturization, which can significantly increase the number of parameters and ligands that can be acquired.
• miniaturized DNS technology makes it possible to analyze more than 10 ⁇ parameters per cm 2 , thus achieving a miniaturization level of less than 10 ⁇ m 2 / parameter on one chip.
• acceptor molecules - which interact with the ligands - on a chip require that for each test set the positioning procedure on the carrier material must be repeated in the same way for all acceptor molecules, in order to to ensure regular arrangement - the so-called array - on the carrier.
• microparticles as a DNA array.
• Such a microparticle array is based on the fact that several suspensions of microparticle populations which have different, discrete fluorescent labels are conjugated with specific acceptor molecules in each case (Lackner et al, 1999, Medgen 11, pp. 16-17). After the conju- gation of the acceptor molecules, the individual suspensions of the different microparticle populations are mixed and an aliquot of the mixture is added to the sample solution, so that particles from each suspension are mixed in the reaction mixture. The ligands to be detected from the sample solution then bind ligand-specifically to the corresponding acceptor molecules and thus always only to discrete microparticles of a certain population.
• a receptor fluorescent dye is bound to the ligands, the emission wavelength of which differs sufficiently from the emission wavelength of the fluorescent dye for labeling the microparticles.
• the fluorescence for identifying the particles, as well as the reporter fluorescence of the ligands bound to the particles, is then analyzed in the flow cytometer.
• microparticles which contain combinations of fluorescent dyes and which can be used for different detection methods.
• the combination of the fluorescent dyes allows the targeted influencing of the excitation and emission wavelength via the energy transfer between different, polymerized dyes.
• the invention is therefore based on the object of providing an efficient method and a test kit which allow a shorter measuring time and a higher sensitivity, are inexpensive and can be used in routine operation.
• the present invention solves this technical problem by providing a method for detecting analytes, the method comprising the following steps: fluorescent labeling of at least one microparticle population, the microparticle population at least one fluorescent dye which serves as coding fluorescence and at least one fluorescent dye which serves as reference fluorescence, comprises binding and / or conjugation of acceptor molecules to the microparticle population, immobilization of the microparticle population on a support, incubation of the microparticle population with the sample to be examined, labeling of the analyte (s) with at least one fluorescent dye which serves as reporter fluorescence, and Detection of the analyte (s) by comparison the fluorescence of the microparticle population with the reporter fluorescence.
• the method according to the invention comprises several steps which can be modified in their sequence.
• the analytes to be labeled with a fluorescent dye which serves as reporter fluorescence before or after binding.
• microparticles in the sense of the invention are heterogeneous and / or homogeneous fractions of microscopic particles with a size of 1-500 ⁇ m, in particular 1-100 ⁇ m, preferably 1-10 ⁇ m.
• the microparticles can contain organic and / or inorganic components.
• the microparticles can be, for example, polymers which, after emulsification or interfacial polymerization, are deposited on the material to be enclosed, for example a fluorescent dye.
• the microparticles can consist of polystyrene or polyphosphoric acid, polyvinyl or polyacrylic acid copolymers.
• microparticles consist of oxidic ceramic particles, such as silicon dioxide, titanium dioxide or other metal oxides.
• oxidic ceramic particles such as silicon dioxide, titanium dioxide or other metal oxides.
• crosslinked polypeptides, proteins, nucleic acid, macromolecules, lipids, for example as vesicles and the like are also microparticles in the sense of the invention.
• the production of microparticles is disclosed for example in the patents US 6,022,564, 5,840,674, 5,788,991 and 5,7543,261.
• a microparticle population in the sense of the invention is understood to mean microparticles which are related to the The fluorescent dye or fluorescent dyes are the same.
• a microparticle population can consist of microparticles which have been labeled with a red, yellow or blue fluorescent dye and / or with fluorescent dyes whose fluorescence lifespan differs.
• a microparticle population can also be defined by the ratio of different fluorescent dyes.
• a microparticle population can then include, for example, all microparticles whose fluorescent marking consists, for example, of green and red fluorescent dye in a 1: 1 ratio.
• the microparticles of a microparticle population have at least one fluorescent dye which serves as coding fluorescence and at least one further fluorescent dye which serves as comparative fluorescence.
• the coding fluorescence is used to analyze the microparticles.
• the microparticles can be labeled with different fluorescent dyes or with different intensities of the fluorescent dyes. This creates discrete microparticle populations that can be identified with the help of detectors.
• the microparticles have at least one further fluorescent dye, which serves as reference fluorescence. With the help of the comparative fluorescence, it is possible to effectively reference the coding fluorescence. By equipping the microparticles with the comparison fluorescence and a coding fluorescence, the fluorescence signals can be related to one another and measurement errors can thus be compensated for.
• Fluorescent dyes that are used to label the microparticles are all substances that can send detectable luminescence signals. However, dyes can also be used are used which emit X-rays or have phosphorescence. Fluorescent dyes in the sense of the invention are all gaseous, liquid or solid inorganic and / or organic compounds which are characterized in that after excitation they release the absorbed energy in the form of radiation of the same, longer or shorter wavelength. This means that inorganic or organic luminescent pigments or quantum dots can also be used as fluorescent dyes in the sense of the invention. However, it can also be provided that the microparticles are designed in such a way that they have their own fluorescence, or both their own fluorescence and a non-own fluorescent label.
• microparticles can be generated, for example, in that the microparticles contain the mineral fluorite.
• non-proprietary fluorescent dyes for coding and / or comparative fluorescence are: dansyl chloride, fluorescein isothiocyanate, 7-chloro-4-nitrobenzoxadiazole, pyrenebutyrylacetic acid anhydride, N-iodoacetyl-N ⁇ - (5-sulfonic acid-1-naphthyl) - diamine, l-anilinonaphthalene-8-sulfonate, 2-toluidinonaphthalene-6-sulfonate, 7- (p-methoxybenzylamino) 4-nitro-benz-2-oxa- 1,3-diazole, formycin, 2-aminopurine ribo- nucleoside, etheno-adenosine, benzoadenosine, ⁇ - and ⁇ -parina
• transition metal complexes which contain the following substances: ruthenium (II), rhenium (I) or osmium and iridium as the central atom and diimine ligands; phosphorescent propyrins with platinum, palladium, lutetium or tin as the central atom; phosphorescent complexes of rare earths such as europium, dysprosium or terbium; phosphorescent crystals like Ruby, Cr-YAG, alexandrite or phosphorescent mixed oxides such as magnesium fluorogermanate or cadmium selenite crystals, fluorescein, aminofluorescein, aminomethylcoumarin, rhodamine, rhodamine 6G, rhodamine B, tetramethylrhodamine, ethidium bromide and / or acridine orange.
• transition metal complexes which contain the following substances: ruthenium (II), rhenium (I) or o
• fluorescent dyes for coding fluorescence e.g. the following substances are used:
• the fluorescent dyes can, for example, be polymerized in during the production of the microparticles or subsequently co-immobilized on the microparticles.
• the fluorescent dyes can, for example, be introduced directly into a solvent for the microparticles during the production of the microparticles. By polymerizing the fluorescent dyes, it is possible to precisely determine the amount of fluorescent dyes bound to the microparticles.
• the fluorescent dyes can either be present in copolymerized form in such a way that they are largely inert or else interact with the analytes.
• the incorporation of the fluorescent dyes in a sol-gel glass as microparticles with subsequent boiling, pulverizing and dispersing the glass is possible.
• the dye can be dispersed as a sensitive layer, for example in the form of the coating on the outside of the microparticles. This can be done, for example, by covalently or electrostatically binding the fluorescent dyes to the surface of the microparticles.
• hydroxyl groups, amphiphilic electrolytes, phospholipids and ionic components can be used to bind fluorescent dyes to the surface of the microparticles.
• the fluorescence-labeled microparticles conjugate or bind with acceptor molecules.
• specific acceptor molecules are coupled to each population of microparticles.
• the acceptor molecules can be functional groups, such as amino groups, carboxyl groups, tiol groups, hydroxyl groups or else epitopes, paratopes, carbohydrates, lectins or oligonucleotide or polynucleotide sequences.
• Epitopes that are used for immobilization can be, for example, antigenic determinants that interact with the antigen-binding part of an antibody or with a receptor.
• Paratopes in the sense of the invention can be, for example, the parts of an antibody that specifically interact with antigenic structures.
• the acceptor molecules can bind covalently, non-covalently, by ionic binding or other interactions to the respective microparticle population.
• the microparticle populations are immobilized on a carrier.
• the immobilization places the microparticles in a state limited by the reaction space.
• immobilization is understood to mean all methods which lead to restriction of the mobility of the microparticles in a biological, chemical or physical way.
• the carrier on which the microparticles are immobilized can be, for example, glass plates, membranes, braids and / or fibrils.
• the microparticles can be immobilized on a support directly or via spacers.
• Spacers in the sense of the invention are all spacers which can, for example, form a short carbon chain between microparticles and carrier.
• hydroxylated chains can be used to avoid specific hydrophobic interactions.
• the acceptor molecules have the properties required for this, such as molecular charge, chemically modifiable groups and / or immune or nucleic acid, hybridization affinities, inter alia, by immobilizing the microparticles with the aid the acceptor mole Immobilization with the aid of the spacers does not necessarily have to be carried out.
• the microparticles immobilize on the surface of the microparticles on the carrier via binding sites.
• At least one microparticle population is incubated with the sample to be examined.
• the incubation of immobilized microparticles and the sample to be examined makes it possible for analytes from the sample to interact with the acceptor molecules which are bound to the microparticles.
• the acceptor molecules are bound antibodies
• the analytes for example antigenic structures, can bind to them. Since the acceptor molecules are bound to the microparticles, this enables the analytes to bind to the microparticles via the acceptor molecules.
• reaction conditions can be created during the incubation of the microparticle population with the sample to be examined, which enable efficient interaction between the analytes and the microparticle population; such reaction conditions can be, for example, an elevated temperature.
• reaction conditions can be, for example, an elevated temperature.
• the number of the microparticle population to be immobilized on the carrier is determined in particular by the number of acceptor molecule specificities that are required for the characterization of the analytes.
• the desired suspensions are mixed, for example, and small aliquots of the mixture are pipetted onto the carrier using a dispenser.
• microparticles sedate in the droplet and contact the surface of the support 1 - 1,000,000, in particular 1 - 100,000, preferably 1 - 10,000 and particularly preferably 1 - 1,000 or fewer microparticles of a population being bound to the support in a randomly distributed manner. NEN. Drying of the drop on the carrier must be prevented in particular if the drying of the acceptor molecules has an adverse effect on the desired binding of the analytes.
• the analytes are labeled with at least one reporter fluorescence. It is of course possible to label the analytes before or after binding to the acceptor molecules. Fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, Texas red, 7-amino-4-methyl-coumarin-3-acetic acid, phycoerythrin and / or cyanines and others, or antibody-conjugated fluorescent particles which are used, for example, with the aid of antibodies on the analyte, can be used as fluorescent molecules , For example, it is possible to label the analytes directly with a fluorescent dye.
• fluorescence-labeled antibodies or other marker-carrying structures can be dispensed with.
• the ligands can be directly labeled in particular by fluorescent dyes which emit a fluorescent signal or specifically quench the fluorescence of other markers.
• the analytes can also be enzyme-labeled. Examples of enzyme-labeled molecules are horseradish peroxidase, alkaline phosphatase and / or glucose oxidase.
• the analyte (s) is detected by comparing the fluorescence of the microparticle population with the reporter fluorescence.
• the intensities of the fluorescence of the microparticle population and the analyte (s) can be compared in a fluorescence spectrometric determination such that in particular both the identity of the analyte-projecting microparticle population and the number of bound Analytes can be analyzed. This makes it possible, for example, to say which analytes have bound to certain discrete microparticle populations and in what number.
• the parameters of the comparison fluorescence are not influenced by the analytes, while, for example, the intensity of the coding fluorescence can change depending on the respective concentration of the analytes.
• the signal intensity of the microparticle population can thus be characterized by internal referencing, in particular without the need for a further excitation source for the fluorescence or a second detector for determining the fluorescence.
• the reference variable for internal referencing is determined on the basis of the fluorescence intensity and / or the time course of the fluorescence response of the comparison fluorescence. It is advantageous if the coding fluorescence and the comparison fluorescence absorb light in the same wavelength range and can thus be excited with the same excitation source. The emission spectra are advantageously in the same spectral range.
• the method allows the comparative fluorescence not to have a specific reaction for the molecules to be detected, but to provide a constant signal for referencing.
• both the comparison fluorescence and the coding fluorescence interact with the analytes.
• neither the comparison fluorescence nor the coding fluorescence interact with the analytes in such a way that the fluorescence signals change.
• the fluorescent dyes for the coding fluorescence and the comparative fluorescence to be excited simultaneously and jointly by a source and to be jointly detected by a detector. It can also be provided be that the comparison fluorescence serves to excite the reporter fluorescence.
• microparticles bind to micro test plates, glass plates, flexible membranes, braids or fibrils, in particular made of polypropylene and / or nitrocellulose, glass and / or polyvinylidene fluoride (PVDF).
• micro test plates e.g. Microtiter plates are used.
• microtest plates have dimensions that permit their use in numerous laboratory routines. For example, numerous fluorescence measuring devices, such as fluorescence microscopes and the like, are designed in such a way that microtest plates can be used as a standard.
• microtiter plates for example microtiter plates, petri dishes, multiple dishes, multi-dishes, tub dishes and other culture vessels and slides, therefore advantageously also enables the use of the available laboratory means and devices for incubating, freezing, lyophilizing and similar laboratory devices in clinical or research laboratories ,
• microtiter plates with transparent, non-fluorescent flat bottoms can preferably be used as micro test plates.
• analytes are detected simultaneously by loading the analytes with individual A-acceptor molecules via discrete microparticle populations. For example, various laden Mi ⁇ can be marked kropismepopulationen to them subsequently ⁇ hd to mix. Aliquots of the microparticle population mixture are then contacted on a carrier in order to immobilize the microparticles on the carrier. The carrier is then advantageously incubated with the sample that the analytes bind to the acceptor molecules which are assigned to the respective microparticle population.
• the parallel acquisition of several parameters by the simultaneous detection of different analytes allows the characterization of several analytes with little material and time.
• the analytes are bound via a linker molecule.
• the linker molecule can be bound, for example, covalently or non-covalently to the analyte.
• the linker molecule can modulate the mobility of the analytes so that the signal emitted by the analyte or by the fluorescent dye bound to the analyte can be efficiently detected.
• the analytes are specifically labeled with different fluorescent dyes.
• the fluorescent dyes can differ by the color of the fluorescence, the fluorescence lifetime or by the intensities.
• the marking with fluorescent dyes in different intensities can advantageously result in a discrete microparticle population which can be detected, for example, in the fluorescence microscope.
• the possible number of discrete populations depends in particular on the available dyes and techniques for labeling the microparticles and on the number of distinguishable colors in the defection measuring device. For example, it is advantageously possible to produce approximately 60 to 100 different discrete microparticle populations with two colors. The number of the population can be increased by a more precise determination of the intensities or by an additional color to approx.
• the analytes are labeled with a uniform fluorescent dye.
• the analytes can be analyzed, for example, by assigning them to discrete populations by labeling the microparticles.
• the fluorescent dyes and / or enzymes are in monomeric and / or polymeric form.
• the fluorescent dyes can be, for example, inorganic compounds, such as compounds of the rare earth metals or, for example, uranium compounds or organic compounds.
• chromogenic substrates which in particular are chemically luminescent, instead of being marked by fluorescent substrates.
• fluorescent microparticles can also bind to the analytes.
• different antibodies are detected in a serological sample (see FIG. 1).
• Different colored fluorescent microparticles are conjugated with different antigens as acceptors and immobilized on a carrier via the antigens.
• antibodies advantageously bind as analytes from a patient's serum to the antigens for which they are specific.
• the bound antibodies are detected with the help of a secondary antibody that has reporter fluorescence.
• the fluorescence of the microparticles and the reporter fluorescence, in particular for each pixel of a microtiter plate cavity, are measured.
• the invention also includes a test kit, the test kit comprising at least one fluorescence-labeled, immobilized micro- particle population that can bind with specific acceptor molecules.
• the immobilized microparticles comprise at least two fluorescent dyes which differ in terms of their spectral properties and / or their fluorescence lifetime, one dye being used for coding fluorescence and one dye being used for the comparative fluorescence.
• the comparison fluorescence serves to reference the coding fluorescence.
• the signals of the coding fluorescence can advantageously be set in relation to the comparison fluorescence, and measurement errors can thus be compensated for.
• the reference fluorescence can be used to reliably determine, for example, whether one or more microparticles are in the measuring field.
• Analytes to be examined can also have, for example, a reporter and a reference fluorescence.
• the ratio of coding fluorescence and reference fluorescence can advantageously be detected more precisely by the comparison fluorescence.
• the test kit can advantageously achieve a measurement accuracy which, for example, meets the requirements of routine clinical practice.
• the test kit can be constructed in such a way that, on account of the immobilized microparticles, the fluorescence is evaluated with fluorescence scanners and / or fluorescence microscopes, which, for example, permits high measurement accuracy and speed of the entire process of evaluating the fluorescence, for example compared to flow cytometers.
• the test according to the invention may be constructed so that there are microparticles and acceptor molecules in solid or dissolved in different reaction vessels and the fluorescent dyes and reagents for Property ⁇ billeiter also be stored separately.
• the immobilization and fluorescent labeling of the microparticle population and the binding of the acceptor molecules to the microparticles are carried out in such a way that the immobilized and fluorescence-labeled microparticle population with the bound acceptor molecules are present in a reaction vessel.
• the sample to be examined is then placed in this vessel.
• it is also possible to set up the test in such a way that all the reagents required for the detection of the analyte are already present in one reaction vessel.
• test kit it is advantageously possible to analyze biological and / or chemical samples.
• biological samples such as serum
• molecular parameters for the characterization of complex biomedical conditions such as the immune status or the genetic predisposition for certain diseases or the recording and influencing of the expression can be recorded.
• immobilizing the microparticle population for example, a sample that has only a small number of analytes to be examined or competitive analytes can be characterized in a very short period of time.
• the invention also includes the use of fluorescence-labeled microparticle populations conjugated to specific acceptor molecules, such as e.g. human or animal antibodies against infectious agents, antigens, autoantigens and allergens, pharmacologically important binding sites in proteomes, genomes and other nucleic acids, e.g. Hormone receptors, pharmaceutical binding parts, peptide, carbohydrate and DNA binding sites and / or for carrying out expression analyzes of important genes and their products, such as e.g. Tumor proteins, HLA antigens and for the analysis of single nucleotide polymorphisms and mutations.
• specific acceptor molecules such as e.g. human or animal antibodies against infectious agents, antigens, autoantigens and allergens, pharmacologically important binding sites in proteomes, genomes and other nucleic acids, e.g. Hormone receptors, pharmaceutical binding parts, peptide, carbohydrate and DNA binding sites and / or for carrying out expression analyzes of important genes and their products,
• the advantages of the method and the test kit according to the invention are, for example, that the immobilization of the micropar- particle populations a reduction in the number of particles is possible. This advantageously leads to an economical use of the acceptor molecules in comparison to flow cytome-fresh methods.
• the detection of the bound analytes via, for example, fluorescent microparticles, quantum dots or luminescent pigments leads to sensitivity down to the single molecule range.
• the 3D structure, in particular of porous particles achieves a high and constant acceptor molecule density, which also contributes to increasing the sensitivity and reproducibility.
• the number of microparticles per sample and the duration of the measurement process of the microparticles can thus be reduced, the sensitivity of the measurement process being increased.
• the use of the comparative fluorescence makes it particularly easy to recognize the measuring fields in which only one microparticle has been immobilized, which is particularly important for the automatic evaluation.
• microparticles By immobilizing the microparticles on standardized supports, such as microtiter plates or slides, existing laboratory routines, such as ELISA machines, can be used, for example.
• Microparticle population A comparative fluorescence: aminocoumarin coding fluorescence: 100% aminofluorescein
• Microparticle population B comparative fluorescence: aminocoumarin coding fluorescence: 50% aminofluorescein
• Microparticle population C comparison fluorescence: aminocoumarin coding fluorescence: 0% aminofluorescein
• Bacterial protein mixtures from Borrelia burgdorferi were coupled to microparticle population A, from Yersinia enterocolitica to microparticle population B, and from Chlamydia trachomatis to microparticle population C by carbodiimide coupling. To do this:
• the breads washed three times in PBS and taken up in 1 ml PBS and aliquoted 50 ⁇ l and frozen at -20 ° C. until further use.
• microparticles of the three populations used are immobilized by drying at 45 ° C.
• the cavities are then rinsed three times with PBS + 0.1% Tween 20 (PBS-T). Human sera are then pipetted in a dilution of 1: 100 in PBS-T into the prepared cavities of the microtest plate and incubated for 1 h at room temperature.
• the cavities are then rinsed three times with PBS + 0.1% Tween 20 (PBS-T) and with goat-anti-human-IgG-anti-serum-phycoerythrin conjugate, which was diluted 1: 100 in PBS-T, for Incubated for 2 h at room temperature. After rinsing three times with PBS-T, the fluorescence ⁇ takes place zenzaustechnisch with a fluorescence microscope Axiophot (Zeiss).
• the immobilized microparticles are with a B / W CCD camera using optical filter pairs for the following emission and absorption wavelengths 390nm / 441 n, 480 nm / 520 nm and 480 nm / 578 nm photographed in succession.
• the evaluation takes place in that a data reduction takes place and only those measuring fields that have a reporter fluorescence intensity above the defined threshold value are included in the evaluation.
• a second data reduction / error comparison is achieved in that all measurement errors that are 20% above and 50% below the comparison fluorescence are excluded from the calculation.
• the quotient of the intensity of the coding fluorescence and the comparison fluorescence provides information about which microparticle population is involved. Quotients that deviate from the mean of a class by more than 10% mean that the measuring field is excluded from the calculation.
• the reporter fluorescence from 20 measuring fields is divided by the associated comparison fluorescence. The resulting quotients of the 20 measuring fields are averaged. They are proportional to the amount of bound human IgG that has specifically bound to the bacterial antigens of this microparticle population.
• Microparticles conjugated with antigen A (yellow) as acceptor molecule Reference fluorescence: yellow; Coding fluorescence: blue (intensity 20%)
• Microparticles conjugated with antigen B (brown) as acceptor molecule Reference fluorescence: yellow; Coding fluorescence: blue (intensity 40%)
• Microparticles conjugated with antigen C (white) as acceptor molecule Reference fluorescence: yellow; Coding fluorescence: blue (intensity 60%)
• Antibody as analyte from the patient serum which is specific to antigen r -.
• A binds

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